This project will use the regenerating caudal fin of the zebrafish as a model system to test the hypothesis that the loss of regenerative capacity in senescence is caused by genomic instability. The significance of this stems from the fact that although this hypothesis has been popular for many decades, it remains controversial and has not been definitively tested. It is important to do so, because if the hypothesis is correct then health decline with age cannot be prevented, only slowed down. The hypothesis is testable because it makes specific predictions in regard to the effects of age, as well as of conditions that are known to either accelerate or decelerate aging. The first prediction is that loss of regenerative capacity with age is proportional to the loss of genomic integrity. The second prediction is that conditions that accelerate aging will increase genomic instability whereas conditions that extend lifespan will have the opposite effect. The third prediction is that genetic networks that function to promote regeneration early in life will tend to lose functionality and/or reliability later in life. These predictions, and hence our hypothesis, will be tested by way of two specific aims that make innovative use of zebrafish as an experimentally tractiable animal model for regeneration research, and high throughput sequencing to analyze genome-wide gene expression. The first aim is to determine how age, chronically elevated stress signaling (via Cortisol), and treatment with the lifespan-extending drug rapamycin affect regenerative capacity, genomic integrity, and mitochondrial function. Toward that end we will perform quantitative PCR of DNA extracted from caudal fins of young, middle aged, or old zebrafish that are either untreated or subjected to long-term treatments with Cortisol or rapamycin, and measure the change in relative amounts of DNA damage in both mitochondrial and nuclear genomes with respect to age and treatment. In addition, the rate and extent of tailfin regeneration will be quantified in each treatment group, and mitochondrial function assessed in tissue samples from each group. The second specific aim is to determine how age, chronically elevated Cortisol, and rapamycin treatment affect regenerative gene expression. Toward that end we will perform high-throughput sequence analyses of RNA (mRNA-seq and microRNA-seq) extracted from regenerating caudal fins dissected from young, middle aged, or old zebrafish that are either untreated or have been subjected to long-term treatment with Cortisol or rapamycin. These studies will both test our hypothesis, and provide the initial data required to systematically map the genomic regulatory systems underlying the decline of regenerative capacity in senescence.